Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

To better understand telomere length set point control in human stem cells, we generated knockout stem cell lines for TPP1 and contrasted their phenotypes with those of homozygous TPP1 L104A mutant stem cells. This comparison reveals that TPP1 L104A is not a hypomorphic allele but formally establishes TPP1 L104 as a dissociation of function mutant.

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Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0365 ◽

2015 ◽

Vol 370 (1680) ◽

pp. 20140365 ◽

Cited By ~ 19

Author(s):

Maria Rostovskaya ◽

Nicholas Bredenkamp ◽

Austin Smith

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cells ◽

Type I ◽

Pancreatic Progenitors ◽

Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

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Reconstructing human early embryogenesis in vitro with pluripotent stem cells

10.1101/2021.03.12.435175 ◽

2021 ◽

Author(s):

Berna Sozen ◽

Victoria Jorgensen ◽

Meng Zhu ◽

Tongtong Cui ◽

Magdalena Zernicka-Goetz

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Human Development ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Early Embryo ◽

Human Stem Cells ◽

Amniotic Cavity ◽

Early Human

ABSTRACTUnderstanding human development is of fundamental biological and clinical importance. Yet despite its significance, insights into early developmental events in humans still remain largely unknown. While recent advances show that stem cells can mimic embryogenesis1–9 to unravel hidden developmental mechanisms, a stem cell-based model of early human embryogenesis is lacking. Here, we use human extended pluripotent stem cells10to reconstitute early human development in 3-dimensions and recapitulate early embryo-like events. We first perform a systematic characterisation to reveal unique signalling requirements for building the human pre-implantation blastocyst. Further, we show that these in vitro stem cell-derived blastocyst-like structures are able to undertake spatiotemporal self-organisation to mimic peri-implantation remodelling in which a polarised rosette opens up the amniotic cavity within a developing disc. The hallmarks of human early development displayed by this stem cell-based in vitro model mimics features of embryonic day 3 to day 9/10 of natural development. Thus, this platform represents a tractable model system to contribute to the basic understanding of cellular and molecular mechanisms governing early embryonic events in humans and to provide valuable insights into the design of differentiation protocols for human stem cells in clinical applications.

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Expression of miRNAs from the Imprinted DLK1/DIO3 Locus Signals the Osteogenic Potential of Human Pluripotent Stem Cells

Cells ◽

10.3390/cells8121523 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1523 ◽

Cited By ~ 1

Author(s):

Laetitia Barrault ◽

Jacqueline Gide ◽

Tingting Qing ◽

Lea Lesueur ◽

Jorg Tost ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Osteogenic Differentiation ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cells ◽

Osteogenic Potential ◽

Human Pluripotent Stem Cell ◽

Stem Cell Lines

Substantial variations in differentiation properties have been reported among human pluripotent cell lines (hPSC), which could affect their utility and clinical safety. We characterized the variable osteogenic capacity observed between different human pluripotent stem cell lines. By focusing on the miRNA expression profile, we demonstrated that the osteogenic differentiation propensity of human pluripotent stem cell lines could be associated with the methylation status and the expression of miRNAs from the imprinted DLK1/DIO3 locus. More specifically, quantitative analysis of the expression of six different miRNAs of that locus prospectively identified human embryonic stem cells and human-induced pluripotent stem cells with differential osteogenic differentiation capacities. At the molecular and functional levels, we showed that these miRNAs modulated the expression of the activin receptor type 2B and the downstream signal transduction, which impacted osteogenesis. In conclusion, miRNAs of the imprinted DLK1/DIO3 locus appear to have both a predictive value and a functional impact in determining the osteogenic fate of human pluripotent stem cells.

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Concise Review; Effects of Antibiotics and Antimycotics on the Biological Properties of Human Pluripotent and Multipotent Stem Cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16999201203214425 ◽

2020 ◽

Vol 16 ◽

Author(s):

Maryam Farzaneh

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Culture Media ◽

Embryonic Stem ◽

Great Promise ◽

Human Stem Cells ◽

Multipotent Stem Cells ◽

The Impact

Abstract:: Human pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the remarkable potential to self-renew and develop into various cell lineages. Human mesenchymal stem cells (MSCs) or multipotent stem cells that are present in various organs can self-renew and differentiate into multiple mesenchymal lineages. Both human PSCs and MSCs hold great promise in cell-based therapies, disease modeling, drug discovery, and regenerative medicine. Human stem cells must be cultured under the optimal conditions to use them in transplantology. Therefore, researchers must ensure the sterility of human stem cell lines. Bacterial contamination is a common problem in laboratories and major precautions are required to detect the types of microorganisms, eliminate, and prevent contamination in cell cultures. Stem cell culture media usually contains antibiotics and antimycotics such as penicillin-streptomycin (pen-strep), gentamicin, and amphotericin B (AmB) to avoid bacterial, fungal, and yeast contaminants. Numerous publications recognized the serious effect of antibiotics and antimycotics on in vitro properties of human stem cells, including proliferation, differentiation, survival, and genetic instability. This review study aimed to understand the impact of routinely used antibiotics and antimycotics such as pen-strep, gentamicin, and AmB on viability, proliferation, and functional properties (differentiation and pluripotency) of human PSCs and MSCs.

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294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab294 ◽

2013 ◽

Vol 25 (1) ◽

pp. 294

Author(s):

J.-K. Park ◽

K.-H. Choi ◽

D.-C. Son ◽

J.-I. Oh ◽

C.-K. Lee

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Embryonic Stem ◽

Embryonic Fibroblasts ◽

Primed State ◽

Exogenous Factors ◽

Stem Cell Lines

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells. This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

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A secreted proteomic footprint for stem cell pluripotency

10.1101/2021.04.14.439804 ◽

2021 ◽

Author(s):

Philip A Lewis ◽

Edina Silajdžić ◽

Helen Smith ◽

Nicola Bates ◽

Christopher A Smith ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Strong Correlation ◽

Proteomic Analysis ◽

Human Stem Cells ◽

Pluripotent State ◽

Non Invasive ◽

Stem Cell Pluripotency ◽

Invasive Method

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the spent medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of 4 E8- against 4 E6- enriched biomarkers provides 16 ratio abundances which are each robustly diagnostic for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to recovery assays.

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High-Resolution Genomic Profiling of Chromosomal Abnormalities in Human Stem Cells Using the 135 K StemArray

Stem Cells International ◽

10.1155/2012/431534 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Aaron M. Elliott ◽

Kristi A. Hohenstein Elliott ◽

Anja Kammesheidt

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Chromosomal Aberrations ◽

Chromosomal Abnormalities ◽

Extended Period ◽

Comparative Genomic ◽

Genetic Profile ◽

Human Stem Cells ◽

Stem Cell Lines

Culturing stem cells for an extended period of time can lead to acquired chromosomal aberrations. Determining the copy number variant (CNV) profile of stem cell lines is critical since CNVs can have dramatic effects on gene expression and tumorigenic potential. Here, we describe an improved version of our StemArray, a stem-cell-focused comparative genomic hybridization (aCGH) microarray, which contains 135,000 probes and covers over 270 stem cell and cancer related genes at the exon level. We have dramatically increased the median probe spacing throughout the genome in order to obtain a higher resolution genetic profile of the cell lines. To illustrate the importance of using the StemArray, we describe a karyotypically normal iPSC line in which we detected acquired chromosomal variations that could affect the cellular phenotype of the cells. Identifying adaptive chromosomal aberrations in stem cell lines is essential if they are to be used in regenerative medicine.

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Selective Ablation of Cochlear Hair Cells Promotes Engraftment of Human Embryonic Stem Cells-derived Progenitors in The Mouse organ of Corti

10.21203/rs.3.rs-132720/v1 ◽

2020 ◽

Author(s):

Hiroki Takeda ◽

Anna Dondzillo ◽

Jessica A. Randall ◽

Samuel P. Gubbels

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Inner Ear ◽

Hair Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Organ Of Corti ◽

Human Stem Cells ◽

Cochlear Hair Cells ◽

Mouse Organ

Abstract Backgroud: Hearing loss affects 25% of the population at ages 60–69 years. Loss of the hair cells of the inner ear commonly underlies deafness and once lost this cell type cannot spontaneously regenerate in higher vertebrates. As a result there is a need for the development of regenerative strategies to replace hair cells once lost. Stem cell-based therapies are one such strategy and offer promise for cell replacement in a variety of tissues. A number of investigators have previously demonstrated successful implantation, and certain level of regeneration of hair and supporting cells in both avian and mammalian models using rodent pluripotent stem cells. However, the ability of human stem cells to engraft and generate differentiated cell types in the inner ear is not well understood. Methods: We differentiate human pluripotent stem cells to the pre-placodal stage in vitro then transplant them into the mouse cochlea after selective and complete lesioning of the endogenous population of hair cells. Results: We demonstrate that hair cell ablation prior to transplantation leads to increased engraftment in the auditory sensory epithelium, the organ of Corti, as well as differentiation of transplanted cells into hair and supporting cell immunophenotypes. Conclusion: We have demonstrated the feasibility of human stem cell engraftment into an ablated mouse organ of Corti.

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Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

10.1101/298778 ◽

2018 ◽

Author(s):

John M. Boyle ◽

Kelsey M. Hennick ◽

Samuel G. Regalado ◽

Jacob M. Vogan ◽

Xiaozhu Zhang ◽

...

Keyword(s):

Stem Cells ◽

Telomere Length ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Telomere Maintenance ◽

Interaction Domain ◽

Set Point ◽

New Type ◽

Mutant Cells

AbstractTelomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in set point establishment remains elusive. Here, we characterize the contribution of TPP1 isoforms and residues outside the TEL patch, TPP1’s telomerase interaction domain, to telomere length control. We demonstrate that TPP1L, the longer minor isoform of TPP1, can partially compensate for loss of the more abundant shorter isoform, TPP1S. Both TPP1S knockout and complete TPP1 knockout cell lines (TPP1 KO) show telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres while TPP1 KO cells do not and die. We compare these phenotypes with that of TPP1L104A/L104A mutant cells that like the TPP1S KO have short stable telomeres. In contrast to TPP1S KO, TPP1L104A/L104A cells respond to increased telomerase. However, TPP1L104A/L104A’s sensitivity to shelterin-mediated feedback is altered, revealing TPP1L104A/L104A as a new type of shelterin mutant with aberrant set point regulation.

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Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Cell Research ◽

10.1038/s41422-021-00592-9 ◽

2021 ◽

Author(s):

Minglei Zhi ◽

Jinying Zhang ◽

Qianzi Tang ◽

Dawei Yu ◽

Shuai Gao ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Biological Research ◽

Marker Genes ◽

Stem Cell Lines ◽

Cloned Gene

AbstractPig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

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